Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe₂SO₄, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10^–10–10^–4 M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10^–6 M, whereas LDH release was inhibited at concentrations of 10^–6–10^–4 M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Lipid peroxidation induced by bolesatine,a toxin ofBoletus satanas: Implication in m5dC variation in Vero cells related to inhibition of cell growth

Bolesatine, a glycoprotein fromBoletus satanas Lenz, has previously been shown to be mitogenic in rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibited protein synthesisin vitro and in severalin vivo systems. The low concentrations (1–10 ng/ml) of bolesatine were shown to activate protein kinase C (PKC)in vitro (cell-free system) and in Vero cells. In the same time, Vero cells significantly proliferated when incubated with bolesatine concentrations ranging from 1 to 10 ng/ml; the DNA synthesis increased by 27–59% as referred to the control, and InsP3 release increased in a concentration-dependent manner, up to 142%. At higher concentrations, 1–10 g in cell-free systems, bolesatine inhibits protein synthesis by hydrolyzing the nucleoside triphosphates GTP and ATP.In the present work, the implication of other toxic mechanisms, such as lipid peroxidation and active radical production, was investigated in relation to inhibition of cell growth, whereas possible modifications of the ratio m⁵dC/dC+m⁵dC were determined in order to correlate with the biphasic action of bolesatine in Vero cells.Low concentrations of bolesatine up to 10 ng/ml do not increase malonaldehyde (MDA) production, while they induce hypomethylation (5.2% as compared to 7.1%). Higher concentrations (above 20 ng/ml) increase MDA production, from 58 ng/mg of cellular proteins to 113 ng/mg at a concentration of 50 ng/ml, for example, and induce hypermethylation in Vero cell DNA. It is concluded that low concentrations of bolesatine that are proliferative induce hypomethylation, which could be one of the pathways whereby bolesatine induces cell proliferation. Higher concentrations which enhance lipid peroxidation also induce hypermethylation. These mechanisms could be at least partly implicated in the pathway whereby bolesatine induces cell death.Abbreviations MDA malonaldehyde - TBA thiobarbituric acid     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes

Polyunsaturated fatty acids (PUFA) are vulnerable to peroxidative attack. Protecting PUFA from peroxidation is essential to utilize their beneficial effects in health and in preventing disease. The antioxidants vitamin E, t-butylhydroxy toluene (BHT) and t-butylhydroxy anisole (BHA) inhibited ascorbate/Fe²⁺-induced lipid peroxidation in rat liver microsomes. In addition, a number of spice principles, for example, curcumin (5–50 µM) from turmeric, eugenol (25–150 µM) from cloves and capsaicin (25–150 µM) from red chillies inhibited lipid peroxidation in a dose-dependent manner. Zingerone from ginger inhibited lipid peroxidation at high concentrations (> 150 µM) whereas linalool (coriander), piperine (black pepper) and cuminaldehyde (cumin) had only marginal inhibitory effects even at high concentrations (600 µM). The inhibition of lipid peroxidation by curcumin and eugenol was reversed by adding high concentrations of Fe²⁺.     

Effects of phenylalanine and its deaminated metabolites on Na+, K+-ATPase activity in synaptosomes from rat brain

The effects of phenylalanine (PHE) and its deaminated metabolites phenylpyruvate (PHP), phenyllactate (PHL) and phenylacetate (PHA) on sodium and potassium activated adenosinetriphosphatase (Na⁺, K⁺-ATPase) in synaptosomes from rat brain were investigated. At very low concentrations (5–10 M), PHE, PHL and PHA inhibited the activity, while PHP stimulated the activity. At intermediate concentrations (50–100 M), all compounds had no effect, but at higher (0.5–1.0 mM) concentrations they inhibited the enzyme activity. Thus all the compounds tested showed a biphasic effect on the enzyme activity. Hydroxylamine inhibited the Na⁺, K⁺-ATPase activity when present alone; simultaneous addition of hydroxylamine and PHE, however, eliminated the inhibitory effects of each other. Reversal of mutual inhibition also occurred in the presence of hydroxylamine and very low (5–10 M) concentrations of PHL or PHA. The inhibitory effects of PHE at all concentrations, and of PHL or PHA at low concentrations, were also eliminated in the presence of EGTA. The data indicate that inhibition of brain membrane Na⁺, K⁺-ATPase by PHE and by low concentrations of PHL and PHA may involve metal ions, but that the inhibition by high concentrations of these metabolites must occur by a different mechanism. Since Na⁺, K⁺-ATPase plays a central role in neuronal function, and the presence of excess PHE and its deaminated metabolites occurs in brain tissue under conditions of experimentally induced hyperphenylalaninemia and genetic phenylketonuria, the neurologic impairment in experimental and genetic PKU may in part be related to the deleterious effects of these compounds on brain ATPase.     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

Lifetable demography of four cladoceran species in relation to algal food (Chlorella vulgaris) density

Algal food density is known to influence life history variables of cladoceran species. It is not, however, well established whether both littoral and planktonic cladocerans show similar trends when exposed to increasing food concentrations. In the present work, we studied the life table demography of four cladoceran species (Ceriodaphnia cornuta, Moina macrocopa, Pleuroxus aduncus and Simocephalus vetulus) in relation to three algal food concentrations (low: 0.5 × 10⁶, medium: 1.5 × 10⁶ and high: 4.5 × 10⁶ cells ml^–1 of Chlorella vulgaris) (in terms of carbon content, these were equivalent to 0.15, 0.45 and 1.35 g ml^–1, respectively) at 25 °C. In general, for all the tested cladoceran species, values of average lifespan, gross reproductive rate, net reproductive rate, generation time and the rate of population growth were higher at lower food concentrations. Furthermore, high food concentration resulted in a negative population growth rate (mean ± standard error: –0.091 ± 0.026) for P. aduncus. The highest population growth rate (0.602 ± 0.014) was recorded for M. macrocopa at low food density. S. vetulus had the longest average lifespan (40 ± 1 d) while M. macrocopa had the lowest (5 ± 1 d). C. cornuta showed better performance at medium food concentration. We conclude that among the algal concentrations used here, 0.5 × 10⁶ – 1.5 × 10⁶ was beneficial not only to the planktonic species but also to the littoral P. aduncus and S. vetulus while 4.5 × 10⁶ cells ml^–1 was unsuitable for all the cladocerans tested.     

Seedling responses of three Australian tree species to toxic concentrations of zinc in solution culture

A frequently desired outcome when rehabilitating Zn toxic sites in Australia is to establish a self-sustaining native ecosystem. Hence, it is important to understand the tolerance of Australian native plants to high concentrations of Zn. Very little is known about the responses of Australian native plants, and trees in particular, to toxic concentrations of Zn. Acacia holosericea, Eucalyptus camaldulensis and Melaleuca leucadendra plants were grown in dilute solution culture for 10 weeks. The seedlings (42 days old) were exposed to six Zn treatments viz., 0.5, 5, 10, 25, 50 and 100 M. The order of tolerance to toxic concentrations of Zn was E. camaldulensis> A. holosericea> M. leucadendra, the critical external concentrations being approximately 20, 12 and 1.5 M, respectively. Tissue Zn concentrations increased as solution Zn increased for all species. Root tissue concentrations were higher than shoot tissue concentrations at all solution Zn concentrations. The critical tissue Zn concentrations were approximately 85 and 110 g g^–1 DM for M. leucadendra, 115 and 155 g g^–1 DM for A. holosericea and 415 and 370 g g^–1 DM for E. camaldulensis for the youngest fully expanded leaf and total shoots, respectively. The results from this paper provide the first comprehensive combination of growth responses, critical external concentrations, critical tissue concentrations and plant toxicity symptoms for three important Australian genera, viz., Eucalyptus, Acacia and Melaleuca, for use in the rehabilitation of potentially Zn toxic sites.     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Population growth of some genera of cladocerans (Cladocera) in relation to algal food (Chlorella vulgaris) levels

We studied the patterns of population growth of 7 cladoceran species (Alona rectangula, Ceriodaphnia dubia, Daphnia laevis, Diaphanosoma brachyurum, Moina macrocopa, Scapholeberis kingi and Simocephalus vetulus) using 6 algal densities, viz. 0.05×10⁶, 0.1×10⁶, 0.2×10⁶, 0.4×10⁶, 0.8×10⁶ and 1.6×10⁶ cells ml^–1, of Chlorella vulgaris for 18 – 30 days. In terms of carbon content these algal concentrations corresponded to 0.29, 0.58, 1.16, 2.33, 4.65 and 9.31 g ml^–1, respectively. Cladocerans in the tested range of algal levels responded similarly, in that increasing the food concentrations resulted in higher numerical abundance and population growth rates (r). The peak population densities were (mean±standard error) 71±5; 17.1±0.4, 3.6±0.3, 12.7±1.1, 18.2±2.7, 15.8±1.0 and 10.9±0.02 ind. ml^–1, respectively for A. rectangula, C. dubia, D. laevis, D. brachyurum, M. macrocopa, S. kingi and S. vetulus. In general, the lowest r values were obtained for D. laevis (0.01±0.001) at 0.05×10⁶ cells ml^–1 food level while the highest was 0.283±0.004 for A. rectangula at 1.6×10⁶ cells ml^–1 of Chlorella. When the data of peak population density for each cladoceran species were plotted against the body length, we found an inverse relation, broadly curvilinear in shape. From regression equations between the food level and rate of population increase, we calculated the theoretical food quantity (the threshold level) required to maintain a zero population growth (r = 0) for each cladoceran species, which varied from 0.107 to 0.289 g ml^–1 d^–1 depending on the body size. When we plotted the cladoceran body size against the corresponding threshold food levels, we obtained a normal distribution curve. From this it became evident that for up to 1300 m body size, the threshold food level increased with increasing body size; however, beyond this, the threshold level decreased supporting earlier observations on rotifers and large cladocerans.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Inducible effects of abscisic acid on sporophyte discs from Laminaria japonica Areschoug (Laminariales, Phaeophyceae)

The effects of exogenous abscisic acid (ABA;10^–7–10^–5 M), a knownplantgrowth regulator, on reproduction and growth were investigated by culturingdiscs from sporophytes of Laminaria japonica Areschoug.ABAplays a role in triggering sorus formation, and it was found that sorusformation of discs was fastest in 10^–5 M ABA. Theapplication of 10^–5 M ABA to culturing discs alsosuppressed the expansion of surface area. ABA contents in sorus and vegetativeparts of the sporophyte were determined by bioassay. The mean ABA content insorus parts obtained from sporophytes was 0.222 ± 0.053g equivalent-ABA g wet weight^–1, which wasabout five times higher than the content found in vegetative parts (0.048± 0.009 g equivalent-ABA g wetweight^–1). Taken together these results suggest that sorusdevelopment requires an elevated level of ABA and is associated with decreasingvegetative growth and that the ABA level of the sporophyte may play a crucialrole in reproduction.     

Binding of dantrolene-Na to the ciliated membrane ofParamecium aurelia

Summary Dantrolene-Na is a muscular relaxant which binds to sarcoplasmic reticulum (SR) with high affinity and decreases the availability of Ca²⁺ channels. The binding of fluorescent compounds, dantrolene-Na, nifedipine and chlortetracycline to the ciliary membrane ofParamecium aurelia has been studied. Dantrolene at the concentrations of 1.9 · 10^–5, 3.8 · 10^–5 and 7.9 · 10^–5 M manifested a punctuated binding pattern to the cell membrane. Isolated cilia also bound dantrolene at their basal portion, whereas deciliated cell bodies lost their dotted binding pattern. Chlortetracycline showed a similar but weaker fluorescent staining. Nifedipine treated cells revealed no sign of fluorescent binding to the membrane and was only taken up in food vacuoles.Based on these observations we propose that dantrolene binding regular arrays ofParamecium cell membrane could be identical to granular plaques observed by electron microscope. The possible functioning of these structures as Ca²⁺ reservoirs is also discussed.     

Improvement of cephalomanine production in Taxus chinensis cells by a combination of sucrose and methyl jasmonate

Cell suspension cultures of Taxus chinensis, supplemented with 25 g sucrose l^–1, produced 11 mg cephalomanine l^–1, 21 g biomass l^–1 and 19 nkat geranylgeranyl diphosphate (GGPP) synthase activity g protein^–1. Supplementation of the cultures with 100 M methyl jasmonate (MJA) produced 17 mg cephalomanine l^–1, 6 g biomass l^–1 and 78 nkat GGPP synthase activity g protein^–1. Addition of sucrose and MJA together produced 24 mg cephalomanine l^–1, 18 g biomass l^–1 and 55 nkat GGPP synthase activity g protein^–1.